Project Summary: Project 2 The incidence of types 1 and 2 diabetes is on the rise, which will lead to increased macro- and microvascular complications. Diabetes is a leading cause of peripheral arterial disease (PAD), a significant risk factor for amputations of digits or limbs. To date, there are no effective therapies. The ligands of the receptor for advanced glycation endproducts (RAGE), such as nonenzymatically glycated proteins (AGEs), S100/calgranulins and high mobility group box 1 (HMGB1), accumulate in non-diabetic, but especially in diabetic PAD tissues. In human subjects, RAGE and its ligands are upregulated in cardiovascular disease (CVD) and PAD tissues, in multiple cell types, but especially in monocytes/macrophages (M?s) and endothelial cells. In murine hind limb ischemia (HLI), a model of ischemic injury to the peripheral vascular system by unilateral ligation and excision of the femoral artery (FAL), mice globally devoid of Ager (the gene encoding RAGE) display significant increases in blood flow and angiogenesis in the affected skeletal muscle in diabetes and non-diabetes vs. wild type (WT) mice. In parallel, and surprisingly, Ager deletion increased inflammatory monocyte subsets, macrophage (M?) content and inflammation in affected skeletal muscle. In contrast, in atherosclerotic mice and in myocardial infarct tissue (Project 1), significantly reduced M? tissue content and inflammation accompanied tissue repair, thereby unveiling novel niche-specific forces that regulate RAGE-dependent inflammatory responses. The cytoplasmic domain of RAGE binds to the formin, DIAPH1, which transduces RAGE ligand-stimulated signal transduction; preliminary data show that mice globally devoid of Diaph1 display significant increases in blood flow after HLI vs. WT mice. Further, our novel observation that DIAPH1 binds to Mitofusin2 (MFN2) links RAGE/DIAPH1 to mitochondrial properties and the myriad consequences for tissue homeostasis after ischemia. This Program Project shows for the first time that RAGE, DIAPH1 and M?s co-localize in human atherosclerosis in the coronary artery. We hypothesize that RAGE/DIAPH1/MFN2-specific cues from infiltrating immune cells and/or the cellular microenvironment mediate cell-intrinsic and/or cell-cell cross-talk mechanisms in M?s and in tissue endothelial cells (ECs) in HLI/FAL, which aggravate tissue damage and quell repair. We will employ novel Ager and Diaph1 floxed mice, small molecule antagonists of RAGE-DIAPH1 interaction and state-of-the-art molecular techniques to uncover mechanisms of diabetic PAD and to identify novel therapeutic targets and strategies. Project 2 will work closely with Projects 1 and 3 and the two Cores to achieve these goals.